Method for the enzymatic resolution of the racemates of aminomethyl-aryl-cyclohexanol derivatives

ABSTRACT

The invention relates to a process for the enzymatic cleavage of racemates of aminomethyl-aryl-cyclohexanol derivatives.

The invention relates to a process for the enzymatic cleavage ofracemates of aminomethyl-aryl-cyclohexanol derivatives.

Treatment of chronic and non-chronic states of pain is of greatimportance in medicine. There is a worldwide need for pain treatmentswith a good action for target-orientated treatment of chronic andnon-chronic states of pain appropriate for the patient, by which is tobe understood successful and satisfactory pain treatment for thepatient. This is documented in the large number of scientific workswhich have been published in the field of applied analgesics and basicresearch in nociception in recent years.

Tramadolhydrochloride-(1RS,2RS)-2-[(dimethylamino)methyl]-1-(3-methoxyphenyl)cyclohexanolhydrochloride—is a known therapeutic for treatment of severe pain.Aminomethyl-aryl-cyclohexanol derivatives such as tramadol((1RS,2RS)-2-dimethylaminomethyl-1-(3-methoxy-phenyl)-cyclohexanolhydrochloride) can accordingly have an analgesic action, as well ashydroxylated tramadol derivatives, such as are described e.g. in EP753506 A1, or they can be used as intermediates for the preparation ofsubstances having an analgesic action (such as e.g. 4- or 5-substitutedtramadol analogues, which are described in EP 753 506 A1 or EP 780 369A1). Precisely tramadol occupies a special position among analgesicshaving an action on the central nervous system in as much as this activecompound brings about potent inhibition of pain without the side effectsknown of opioids (J. Pharmacol. Exptl. Ther. 267, 331 (1993)), both theenantiomers of tramadol and the enantiomers of tramadol metabolitesparticipating in the analgesic action (J. Pharmacol. Exp. Ther. 260, 275(1992)).

As can be seen from this, enantiomers can have significantly differentactions, and in many respects it is very important also to be able toseparate racemates into enantiomerically pure forms as intermediates orin respect of approvals under drug legislation.

Enzymatic transformations have since become the basic operations ofpreparative organic chemistry. Numerous industrial processes withenzymatic key steps which now go far beyond enzymatic cleavage ofracemates of amino acids have also become established in the meantime. Amore up-to-date overview of the use of enzymes in the preparation ofbiologically active compounds is given by Roberts and Williamson (S. M.Roberts, N. M. Williamson, Current Organic Chemistry, 1997, volume 1,1–20).

Luana et al. (A. Luna, A. Maestro, C. Astorga, V. Gotor, Tetrahedron:Asymmetry 1999, 10, 1969–1977) describe the enzymatic cleavage ofracemates via transesterification of cyclic α-aminoalcohols usinglipases and vinyl acetate as the acyl donor. This publication is ofimportance since it is shown that substrates with an aminoalcoholfunctionality can be used.

Forró and Fülöp (E. Forró, F. Fülöp, Tetrahedron: Asymmetry 1999, 10,1985–1993, E. Forró, L. Kanerva, F. Fülöp, Tetrahedron: Asymmetry 1998,9, 513–520) describe the enzymatic cleavage of racemates of reducedcyclic Mannich bases of the following type:

-   -   where n=1,2,3 and R′″=alkyl, alkylaryl, cycloalkyl

The authors make reference to tramadol in the introduction, and in theintroductory text refer to the use of these compounds as units forsubstances potentially having an analgesic action.

In the development of enzymatic processes, in addition to the suitableenzyme system, discovery of suitable reaction parameters is decisive forthe success of the process.

Preparation of enantiomerically pure aminomethyl-aryl-hexanolderivatives, in particular 4- or 5-hydroxylated tramadol derivatives,via fractional crystallization of diastereomeric salts, such as e.g.tartrates, dibenzoyltartrates or dobenzoyltartrates, has so far not beensuccessful. Preparative chromatographic processes can be employed onlyin certain cases for providing enantiomerically pure compounds on amultigram scale. Suitable chromatographic conditions of preparativeseparation have also not yet been found.

The object of the present invention was therefore to discover suitableprocesses for enantiomerically pure separation of the enantiomers ofaminomethyl-aryl-hexanol derivatives, in particular 4- or 5-hydroxylatedtramadol derivatives—also on a larger scale.

The invention therefore provides processes for the enzymatic cleavage ofracemates of aminomethyl-aryl-cyclohexanol derivatives of the generalformula I

wherein X is chosen from

-   -   H, F, Cl, Br, I, CF₃, O—S(O₂)—C₆H₄-pCH₃, OR¹⁴ or OC(O)R¹⁴,        wherein R¹⁴ is chosen from        -   H; C₁–C₁₀-alkyl, C₂–C₁₀-alkenyl or C₂–C₁₀-alkinyl, in each            case branched or unbranched and mono- or polysubstituted or            unsubstituted; C₃–C₇-cycloalkyl, saturated or unsaturated            and mono- or polysubstituted or unsubstituted, or a            corresponding heterocyclic radical in which a C atom in the            ring is replaced by N, S or O; alkylaryl or alkylheteroaryl,            saturated or unsaturated and mono- or polysubstituted or            unsubstituted; aryl or heteroaryl, in each case mono- or            polysubstituted or unsubstituted;

-   R³, R⁴ independently of one another are chosen from    -   H, C₁–C₁₀-alkyl, C₂–C₁₀-alkenyl or C₂–C₁₀-alkinyl, in each case        branched or unbranched and mono- or polysubstituted or        unsubstituted; C₃–C₇-cycloalkyl, saturated or unsaturated and        mono- or polysubstituted or unsubstituted, or a corresponding        heterocyclic radical in which a C atom in the ring is replaced        by N, S or O; alkylaryl or alkylheteroaryl, saturated or        unsaturated and mono- or polysubstituted or unsubstituted; aryl        or heteroaryl, in each case mono- or polysubstituted or        unsubstituted;        or

-   R³ and R⁴ together form a C₃–C₇-cycloalkyl, saturated or unsaturated    and mono or polysubstituted or unsubstituted, or a corresponding    heterocyclic radical in which a C atom in the ring is replaced by S,    O or NR¹⁵, where R¹⁵ is chosen from    -   H, C₁—C₁₀-alkyl, C₂–C₁₀-alkenyl or C₂–C₁₀-alkinyl, in each case        branched or unbranched and mono- or polysubstituted or        unsubstituted;

-   R¹ and R² independently of one another are either H or any desired    substituent    and

in each case one of the substituents R⁵ and R⁶ corresponds to H and theother corresponds to OH, characterized in that, depending on the desiredenantiomer of the aminomethyl-aryl-cyclohexanol derivatives of thegeneral formula I

either in reaction alternative I

the racemate of compounds according to formula I is first esterified andthen transformed enzymatically and the enantiomerically pure compoundsformed are separated

or in reaction alternative II

the racemate of compounds according to formula I is transformedenzymatically in the presence of an ester and the enantiomerically purecompounds formed are separated.

These processes utilize in particular the fact that reactionalternatives I and II are to be regarded as complementary processes,since in the enzymatic transformation of the racemic mixture theparticular opposite stereochemistry is induced.

In reaction alternative I, a racemic compound according to formula II

in which the substituent OC(O)R⁷ corresponds to the position of R⁵ or R⁶in formula I and R⁷ is chosen from C₁–C₆-alkyl, unsubstituted or mono-or polysubstituted; as the free base or in the form of its salt, istransformed enzymatically in a solvent with a lipase or esterase and theenantiomerically pure compounds formed, according to formulae III and Ia

where compounds according to formula Ia correspond to compoundsaccording to formula I and the substituent OH corresponds to theposition of R⁵ or R⁶ in formula I, are separated.

It is particularly preferable in reaction alternative I if R⁷ informulae II and III is chloroacetyl, butyl or pentyl.

An esterase, in particular a pig liver esterase, is preferably used asthe enzyme in reaction alternative I.

The preferred solvent in reaction alternative I is an aqueous buffersystem, which preferably has a pH of between 6.0 and 8.0—preferably a pHof between 7.0 and 7.5. It is also favourable here if the solvent is anaqueous buffer system with a physiological pH for the enzyme used. It isparticularly favourable here if one or more organic solvent(s),preferably acetone or butanol, is/are added to the aqueous buffer systemup to a percentage content by volume of between 1 and 50%, preferably 5and 20%, in particular 20%.

It is furthermore preferable, in particular in the case of an aqueousbuffer system, to employ the compound according to formula II as thehydrochloride salt in reaction alternative I.

It is of particular importance here that in the enzymatic hydrolysis ofthe butyric acid ester of 4-hydroxytramadol in particular, but also inother cases according to reaction alternative I—precisely with anaqueous buffer system—the use of the salt, in particular thehydrochloride, and not the base can lead to better results. The base isoften not soluble in a sufficient amount in the aqueous buffer system.It is furthermore particularly remarkable that a significant improvementin the process, in particular also when the hydrochloride is employed,is to be observed if acetone and butanol are added. This particularlyapplies to the rate of reaction. In particular, an addition of acetoneor butanol to the aqueous buffer in an amount of up to between 5 and20%, preferably 20%, of the total volume is often optimum in respect ofselectivity and rate of reaction.

The use of amino-hydrochlorides in enzymatic separations also has nothitherto been described in the prior art.

To prepare the ester of the compounds according to formula II inreaction alternative I, racemic compounds according to formula I

are converted with bases, preferably potassium tert-butylate or sodiumhydride, in a solvent, preferably tetrahydrofuran or dimethylformamide,into the alcoholates and subsequently, with the addition ofcorresponding acid halides, into the racemic esters according to formulaII

in which the substituent OC(O)R⁷ corresponds to the position of R⁵ or R⁶in formula I. The esters according to formula II can preferably beprepared in this way.

In reaction alternative II, a racemic compound according to formula I

employed as the free base or in the form of its salt in a solvent withan ester according to formula IV

wherein, independently of one another, R⁸ denotes C₁–C₆-alkyl,substituted or unsubstituted; and R⁹ denotes H or C₁–C₆-alkyl,substituted or unsubstituted, is transformed enzymatically with a lipaseor esterase and the enantiomerically pure compounds formed, according toformulae V and Ib

wherein compounds according to formula Ib correspond to compoundsaccording to formula I and the substituent OH corresponds to theposition of R⁶ or R⁶ in formula I, are separated.

It is particularly preferable here if, in reaction alternative II, R⁸ inthe esters according to formulae IV and V denotes methyl or ethyl and/orR⁹ according to formula IV denotes H or methyl.

In particular, the ester according to formula IV is preferably vinylpropionate, vinyl acetate or isopropenyl acetate.

A lipase, in particular a lipase from Candida rugosa, Candidacylindracea or Pseudomonas cepacia, is preferably used as the enzyme inreaction alternative II.

It has also proved particularly favourable to use an organic solvent,preferably toluene, as the solvent in reaction alternative II.

A decisive advantage of the process according to the invention by bothreaction alternatives is the easily achievable separation of theenantiomerically pure compounds after conclusion of the enzymatictransformation. The ester/alcohol mixtures here are separated bypH-selective extraction after conclusion of the enzymatictransformation. A chromatographic separation can advantageously beomitted. By establishing a suitable pH, the ester and alcohol can beseparated from one another by extraction, in particular by pH-selectiveextraction, on the basis of sufficiently different log P values.Scaling-up is therefore possible without problems and is particularlyeasy to carry out industrially.

The enzymatic processes found, by both reaction alternatives, arecurrently the only possibility of preparingaminomethyl-aryl-cyclohexanol derivatives, in particular hydroxylatedtramadol derivatives, on a multigram scale with adequate purity of theenantiomers.

Overall, but in particular in the ester cleavage according to reactionalternative I, the conversion can be conducted to up to almost 50%without the selectivity being reduced drastically, as in many comparableenzymatic cleavages of racemates. Over-hydrolysis was not to be observedunder the reaction conditions used.

It is furthermore particularly preferable that the substituents R¹ andR² in the formulae I, Ia, Ib, II, III and V independently of one anotherare chosen from R¹⁰ or YR¹⁰, where Y=C₁–C₁₀-alkyl, C₂–C₁₀-alkenyl orC₂–C₁₀-alkinyl, branched or unbranched and mono- or polysubstituted orunsubstituted, wherein R¹⁰ is chosen from

-   -   H, F, Cl, Br, I, CN, NO₂, C₁–C₈-alkyl, C₂–C₈-alkenyl or        C₂–C₈-alkinyl, in each case branched or unbranched and mono- or        polysubstituted or unsubstituted; C₃–C₇-cycloalkyl, saturated or        unsaturated and mono- or polysubstituted or unsubstituted, or a        corresponding heterocyclic radical in which a C atom in the ring        is replaced by S, O or N; aryl or heteroaryl, in each case mono-        or polysubstituted or unsubstituted;    -   OR¹¹, OC(O)R¹¹, OC(O)OR¹¹, OC(S)R¹¹, C(O)R¹¹, C(O)OR¹¹, C(S)R¹¹,        C(S)OR¹¹, SR¹¹, S(O)R¹¹ or S(O₂)R¹¹, wherein R¹¹ is chosen from        -   H, C₁–C₁₈-alkyl, C₂–C₁₈-alkenyl or C₂–C₁₈-alkinyl, in each            case branched or unbranched and mono- or polysubstituted or            unsubstituted; C₃–C₇-cycloalkyl, saturated or unsaturated            and mono- or polysubstituted or unsubstituted, or a            corresponding heterocyclic radical in which a C atom in the            ring is replaced by S, O or N; alkylaryl or alkylheteroaryl,            saturated or unsaturated and mono- or polysubstituted or            unsubstituted; aryl or heteroaryl, in each case mono- or            polysubstituted or unsubstituted; or    -   NR¹²R¹³, C(O)NR¹²R¹³ or S(O₂)NR¹²R¹³, wherein R¹² and R¹³        independently of one another are chosen from        -   H, C₁–C₁₈-alkyl, C₂–C₁₈-alkenyl or C₂–C₁₈-alkinyl, in each            case branched or unbranched and mono- or polysubstituted or            unsubstituted; C₃–C₇-cycloalkyl, saturated or unsaturated            and mono- or polysubstituted or unsubstituted, or a            corresponding heterocyclic radical in which a C atom in the            ring is replaced by S, O or N; alkylaxyl or alkylheteroaryl,            saturated or unsaturated and mono- or polysubstituted or            unsubstituted; aryl or heteroaryl, in each case mono- or            polysubstituted or unsubstituted;        -   or        -   R¹² and R¹³ together form a C₃–C₇ cycloalkyl, saturated or            unsaturated and mono- or polysubstituted or unsubstituted,            or a corresponding heterocyclic radical in which a C atom in            the ring is replaced by S, O or N;            or

-   R¹ and 2 together form —CH═CH—CH═CH—, wherein the naphthyl system    formed can be mono- or polysubstituted.

The following definitions apply to the complete description of theentire invention described here, and in particular also the sections anddefinitions of radicals presented above, unless expressly definedotherwise.

In connection with alkyl, alkenyl, alkinyl and cycloalkyl or the“corresponding heterocyclic radical”, the term substituted here isunderstood in the context of this invention as replacement of a hydrogenradical by F, Cl, Br, I, NH₂, SH or OH, polysubstituted radicals beingunderstood as radicals which are polysubstituted both on different andon the same atoms, for example trisubstituted on the same C atom, as inthe case of CF₃, or at different points, such as in the case of—CH(OH)—CH═CH—CHCl₂.

Furthermore, —C(O)— denotes

which also applies to —C(S)— or —S(O)— or —S(O₂)—.

The term “C₁–C₈-alkyl” or “C₁–C₁₀-alkyl” in the context of thisinvention denotes hydrocarbons having 1 to 8 or 10 carbon atomsrespectively. Examples which may be mentioned are methyl, ethyl, propyl,isopropyl, n-butane, sec-butyl, tert-butyl, n-pentane, neopentyl,n-hexane, n-heptane, n-octane, n-nonane or n-decane.

The term “C₁–C₁₈-alkyl” in the context of this invention denoteshydrocarbons having 1 to 18 carbon atoms. Examples which may bementioned are methyl, ethyl, propyl, isopropyl, n-butane, sec-butyl,tert-butyl, n-pentane, neopentyl, n-hexane, n-heptane, n-octane,n-nonane, n-decane, n-undecane, n-dodecane, n-dodecane, n-tridecane,n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane orn-octadecane, unsubstituted or mono- or polysubstituted.

The term “C₂–C₁₀-alkenyl” or “C₂–C₁₀-alkinyl” or “C₂–C₁₈-alkenyl” or“C₂–C₁₈-alkinyl” in the context of this invention denotes hydrocarbonshaving 2 to 8 or 2 to 18 carbon atoms respectively. Examples which maybe mentioned are propenyl, butenyl, pentenyl, hexenyl, heptenyl,octenyl, unsubstituted or mono- or polysubstituted, or propinyl,butinyl, pentinyl, hexinyl, heptinyl, octinyl, unsubstituted or mono- orpolysubstituted.

The term C₃–C₇-cycloalkyl in the context of this invention denotescyclic hydrocarbons having 3 to 7 carbon atoms. Examples which may bementioned are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclopentenyl, cyclohexenyl or cycloheptenyl, saturated orunsaturated and substituted or mono- or polysubstituted. A“corresponding heterocyclic radical” here in the context of theinvention is understood as a C₃–C₇-cycloalkyl in which at least one Catom in the ring is replaced by S, O or N. Examples of these which maybe mentioned are pyrrolidine, pyran, thiolane, piperidine ortetrahydrofuran.

The term “aryl” in the context of this invention denotes phenyls,naphthyls or anthracenyls. The aryl radicals can also be fused withfurther rings.

The term “heteroaryl” in the context of this invention denotes aromaticcompounds which are optionally provided with a fused-on ring system andcontain at least one heteroatom from the group consisting of nitrogen,oxygen and/or sulfur. Examples which may be mentioned in this group arethiophene, furan, pyrrole, pyridine, pyrimidine, quinoline,isoquinoline, phthalazine or quinazoline.

The term “alkylaryl” or “alkylheteroaryl” in the context of thisinvention denotes aryls or heteroaryls, where the terms aryl, heteroaryland alkyl have the same meaning as above, which are substituted at leastby C₁–C₆-alkylene and in which bonding is via the alkyl radical.

In respect of “aryl”, “alkylaryl”, “heteroaryl” or “alkylheteroaryl”, inthe context of this invention mono- or polysubstituted is understood asmeaning substitution of the ring system by F, Cl, Br, X, NH₂, SH, OH,CF₃; ═O or ═S; mono- or polysubstituted or unsubstituted C₁–C₆-alkyl,C₁–C₆-alkoxy, C₂–C₈-alkenyl, C₂–C₈-alkinyl; phenyl or benzyl; on one ordifferent atoms.

It is particularly advantageous if R¹ in the formulae I, Ia, Ib, II, IIIand V is R¹⁰, wherein R¹⁰ is chosen from

-   -   H, F, Cl, Br, I, CF₃, NO₂, NH₂; C₁–C₄-alkyl or C₂–C₄-alkenyl,        branched or unbranched and mono- or polysubstituted or        unsubstituted; OR¹¹, C(O)OR¹¹ or SR¹¹, wherein R¹¹ is chosen        from        -   H; C₁–C₄-alkyl, branched or unbranched and mono- or            polysubstituted or unsubstituted; preferably H, CF₃ or CH₃,    -   or S(O₂)NR¹²R¹³, wherein R¹² and R¹³ independently of one        another are chosen from        -   H; C₁–C₄-alkyl, branched or unbranched and mono- or            polysubstituted or unsubstituted;            wherein R¹ is particularly preferably chosen from    -   H, F, Cl, OH, CH₃, C₂H₅, C₂H₃, CF₃, SCH₃, OCF₃, OCH₃, OC₂H₅,        C(O)OCH₃, C(O)OC₂H₅, preferably m-OCH₃.

In particular, the substituent R² in the formulae I, Ia, Ib, II, III andV can be R¹⁰, wherein R¹⁰ is chosen from

-   -   H, F, Cl, Br, I, SCH₃; C₁–C₄-alkyl, C₂–C₄-alkenyl, branched or        unbranched and mono- or polysubstituted or unsubstituted,        preferably CF₃; OR¹¹, where R¹¹ is chosen from C₁–C₄-alkyl,        branched or unbranched and mono- or polysubstituted or        unsubstituted, preferably CH₃;        wherein R² particularly preferably=H.

It is furthermore particularly preferable if X in the formulae I, Ia,Ib, II, III and V is chosen from

-   -   H, F, Cl, OH, CF₃, O—S(O₂)—C₆H₄-pCH₃ or OC(O)R¹² where R¹²=H;        C₁–C₄-alkyl or C₂–C₄-alkenyl, branched or unbranched and mono-        or polysubstituted or unsubstituted,    -   preferably H, F, Cl, OH, O—S(O₂)—C₆H₄-pCH₃, OC(O)R¹², where        -   R¹²=C₁–C₄-alkyl, preferably CH₃;            wherein X is particularly preferably OH, F or Cl, preferably            OH.

It is furthermore a preferred subject matter of the invention if R³ andR⁴ in the formula I, II, III and V independently of one another arechosen from

-   -   C₁–C₄-alkyl, branched or unbranched and mono- or polysubstituted        or unsubstituted, preferably CH₃,        or

-   R³ and R⁴ together form a C₃–C₇-cycloalkyl, saturated or unsaturated    and mono- or polysubstituted or unsubstituted,

Wherein R³ and R⁴ particularly preferably each denote CH₃.

The invention also furthermore provides intermediate products accordingto formula II. The definition of the radicals R¹–R⁴ and X and R⁷mentioned has already been described above, as has also a preferredpreparation process for products according to formula II in the contextof reaction alternative I. The compounds according to formula II arevery suitable analgesics and can also be employed for furtherindications. They are therefore suitable, in the form of theirdiastereomers or enantiomers and their free base or a salt formed with aphysiologically tolerated acid, in particular the hydrochloride salt,for the preparation of a medicament for treatment of pain, in particularmigraine, acute pain and neuropathic or chronic pain, of inflammatoryand allergic reactions, depressions, drug and/or alcohol abuse,gastritis, cardiovascular diseases, respiratory tract diseases,coughing, mental illness and/or epilepsy, and in particular of urinaryincontinence, itching and/or diarrhoea.

The invention is explained below in more detail by examples, withoutlimiting it thereto.

EXAMPLES

The following examples show processes according to the invention.

The following information generally applies in these:

The chemicals and solvents employed were obtained commercially from theconventional suppliers (Acros, Avocado, Aldrich, Fluka, Lancaster,Maybridge, Merck, Sigma, TCI etc.) or synthesized.

Example 1

Preparation of the Carboxylic Acid Eaters of Hydroxy-tramadols

(1SR,3RS,4RS)-Butyric acid3-dimethylaminomethyl-4-hydroxy-4-(3-methoxy-phenyl)-cyclohexyl esterhydrochloride (rac-1)

250 g (0.89 mol)(1RS,2RS,4SR)-2-dimethylaminomethyl-1-(3-methoxy-phenyl)-cyclohexane-1,4-diolrac-2 were suspended in 2,500 ml dried tetrahydrofuran, and 226 gpotassium tert-butylate (2.01 mol) were added in portions, while coolingwith an ice-bath such that the internal temperature did not exceed 30°C. When the addition had ended the mixture was subsequently stirred atroom temperature for a further hour. 127 ml (130.3 g, 1.22 mol) butyricacid chloride were then added, while cooling with an ice-bath, theinternal temperature being between 5 and 10° C. When the addition wascomplete, the mixture was subsequently stirred at room temperature for afurther 15 hours. For hydrolysis, 1,187 ml of a 1 molar aqueous sodiumbicarbonate solution were added dropwise, with renewed cooling with anice-bath. After separation of the phases, the aqueous phase wasextracted twice more with 500 ml ethyl acetate. The combined organicphases were dried over sodium sulfate. After removal of the solvent bydistillation, the residue (277.4 g) was converted into thehydrochloride. For this, the 277.4 g of crude product were dissolved ina solvent mixture comprising 270 ml ethanol and 1,350 ml acetone. Afteraddition of one molar equivalent of trimethylchlorosilane and one molarequivalent of water, the hydrochloride crystallized out. After themixture had been left to stand at 15° C. for 15 hours, the precipitatewas filtered off with suction and, after drying, 273.2 g hydrochloridecould be obtained in a yield of 89%.

Example 2

Preparation of Carboxylic Acid Esters of Hydroxy-tramadols

(1SR,3RS,4RS)-Butyric acid4-dimethylaminomethyl-3-hydroxy-3-(3-methoxy-phenyl)-cyclohexyl esterhydrochloride (rac-3)

Analogously to the preparation of (1SR,3RS,4RS)-butyric acid3-dimethylaminomethyl-4-hydroxy-4-(3-methoxy-phenyl)-cyclohexyl esterhydrochloride rac-1, the ester rac-3 could be obtained in a yield of 85%from(1RS,3SR,6RS)-6-dimethylatinomethyl-1-(3-methoxy-phenyl)-cyclohexane-1,3-diolrac-4.

Example 3

Enzymatic Ester Hydrolysis

Pig liver esterase-catalysed hydrolysis of (1SR,3RS,4RS)-butyric acid3-dimethylaminomethyl-4-hydroxy-4-(3-methoxy-phenyl)-cyclohexyl esterhydrochloride (rac-1)

(−)-(1R,3S,4S)-Butyric acid3-dimethylaminomethyl-4-hydroxy-4-(3-methoxy-phenyl)-cyclohexyl eater((−)-1)and(+)-(1R,2R,4S)-2-Dimethylaminomethyl-1-(3-methoxy-phenyl)-cyclohexane-1,4-diol((+)-2)

72 g (0.19 mol) (1SR,3RS,4RS)-butyric acid3-dimethylaminomethyl-4-hydroxy-4-(3-methoxy-phenyl)-cyclohexyl esterhydrochloride rac-1 were dissolved in 620 ml aqueous phosphate buffersolution pH 7 (Merck, art. no. 1.09439.100), and 140 ml acetone wereadded. After the mixture had been stirred for 10 minutes, a clearsolution was formed. 0.62 g pig liver esterase (Chirazyme E1 from RocheDiagnostics, lyophilisate, 40 units/mg) and 150 ml of a 1 molar aqueoussodium bicarbonate solution were then added in one portion so that a pHof 7.5 was established. The reaction mixture was stirred at roomtemperature for 21 hours. To end the reaction, the buffer system wasextracted twice with 450 ml diisopropyl ether each time and twice with asolvent mixture of diisopropyl ether and diethyl ether in a ratio of 1:1each time, only the ester passing into the organic phase under theseconditions and the hydrolysed alcohol remaining in the aqueous phasebecause of the different logP value (see table 1).

To isolate the ester (−)-1, the combined organic phases were washed oncewith 400 ml of a 1 molar aqueous sodium carbonate solution and driedover sodium sulfate. After removal of the solvent by distillation, 30.4g of crude product (93% of theory) comprising (−)-(1R,3S,4S)-butyricacid 3-dimethylaminomethyl-4-hydroxy-4-(3-methoxy-phenyl)-cyclohexylester (−)-1 were obtained. The crude base ([α]_(D) ²²=−12.0° (c=1.02,methanol)) was taken up in 300 ml of a solvent mixture comprisingethanol and 2-butanone in a ratio of 1:9, and 11.0 mltrimethylchlorosilane and 1.57 ml water were added. 3.6 g (10% oftheory) of the hydrochloride crystallized out with an ee value of 4.8%.After separating off, the mother liquor was concentrated. Afterliberation of the base with sodium carbonate and extraction with ethylacetate, drying over sodium sulfate and removal of the solvent bydistillation, 23.9 g (73% of theory) (−)-(1R,3S,4S)-butyric acid3-dimethylaminomethyl-4-hydroxy-4-(3-methoxy-phenyl)-cyclohexyl ester(−)-1 could be obtained with an ee value of 100% (determined by chiralHPLC).(−)-(1S,2S,4R)-2-Dimethylaminomethyl-l-(3-methoxy-phenyl)-cyclohexane-1,4-diol(−)-2 could be obtained from this in a quantitative yield by alkalineester hydrolysis with potassium hydroxide in ethanol.

For isolation of(+)-(1R,2R,4S)-2-dimethylaminomethyl-l-(3-methoxy-phenyl)-cyclohexane-1,4-diol(+)-2, the aqueous phase of the ester hydrolysis was brought to a pH of5.0 with 2 molar hydrochloric acid. The solution adjusted in this waywas freed from the solvent at a bath temperature of 60° C. under apressure of 650 mbar to 150 mbar. The residue was then brought to a pHof 10.0 with 2 molar aqueous sodium carbonate solution and the mixturewas extracted three times with 100 ml ethyl acetate each time. Thecombined organic phases were dried over sodium sulfate. After removal ofthe solvent by distillation, 26.0 g (100% of theory) crude product couldbe obtained. The crude base was taken up in 270 ml of a solvent mixturecomprising ethanol and 2-butanone in a ratio of 1:9, and 12.2 mltrimethylchlorosilane and 1.73 ml water were added, the hydrochloride of(+)-(1R,2R,4S)-2-dimethylaminomethyl-1-(3-methoxy-phenyl)-cyclohexane-1,4-diol(+)-2 crystallizing out in a yield of 78% (23.1 g) with an ee value of96.3% (according to chiral HPLC) ([α]_(D) ²²=+36.5° (c=1.06 methanol)).

The following table 1 shows the pKa values and logP values of thecompounds 1 and 2.

TABLE 1 pKa values and logP values of the compounds 1 and 2. CompoundCompound 1 2 (ester) (alcohol) pKa value 8.796 9.055 logP value:water/octanol 2.898 1.101 logP value: water/cyclohexane 2.360 −0.632logD value at pH 7.4 water/octanol 1.484 −0.564 logD value at pH 7.40.946 −2.297 water/cyclohexane ΔlogP value at pH 7.4 0.538 1.733 ΔlogDvalue at pH 7.4 0.538 1.733

The following table 2 shows the dependency of the ee value of the esterand alcohol as a function of the reaction time by way of example.

TABLE 2 Dependency of the ee value of the ester and alcohol as afunction of the reaction time (the content and ee value of compounds 1and 2 were determined by means of chiral HPLC): Ester (+) − Ester (−) −Ester % ee value Time in content in content²⁾ content²⁾ of the (−) −hours %¹⁾ (in %) (in %) ester³⁾ 3 91.2 40.8 50.4 10.5 19 68.6 17.7 50.948.4 24 64.4 14.0 50.4 56.6 28 62.2 11.4 50.9 63.4 Alcohol (−) − Alcohol(+) − Alcohol % ee value content in content²⁾ content²⁾ of the (+) − %¹⁾(in %) (in %) alcohol³⁾ 3 8.8 0.4 8.4 91.6 19 31.4 0.5 30.9 96.6 24 35.60.7 35.0 96.2 28 37.8 0.7 37.1 96.5 ¹⁾percentage content of ester oralcohol relates to the total content of ester and alcohol determined inthe reaction mixture; ²⁾the percentage content of enantiomeric esters oralcohols relates to the content of ester and alcohol in the totalmixture ((+) − enantiomer (ester) + (−) − enantiomer (ester) + (+) −enantiomer (alcohol) + (−) − enantiomer (alcohol) = 100%; ³⁾thepercentage ee value was determined according to the following equation:% excess enantiomer − % deficit enantiomer/% excess enantiomer + %deficit enantiomer.

The dependency of the % ee value of the alcohol (+)-2 on the amount ofacetone added is shown in table 3:

TABLE 3 Dependency of the % ee value of the alcohol (+) − 2 on theamount of acetone added (1.5 mmol ester rac-1 as the hydrochloride weredissolved in 5 ml phosphate buffer pH 7.0 (Merck), and 1.2 ml of a 1molar aqueous sodium bicarbonate solution were added; the amount ofenzyme added was 5.0 mg Chirazyme El from Roche Diagnostics; the mixturewas stirred for in each case 19 hours at room temperature; working upwas carried out as described in example 1) Total Total % ee valueAddition ester % ee value alcohol of the of acetone content¹⁾ of thecontent¹⁾ (+) − (ml, %) (in %) (−) − ester²⁾ (in %) alcohol²⁾   0 ml, 0%46.1 90.7 53.9 72.5 0.6 ml, 9% 50.8 97.6 49.2 89.8 1.0 ml, 13.5% 56.885.7 43.3 96.9 1.2 ml, 16% 55.6 94.2 44.4 95.5 ¹⁾the percentage contentof the enantiomeric esters or alcohols relates to the content of esterand alcohol in the total mixture ((+) − enantiomer (ester) + (−) −enantiomer (ester) to (+) − enantiomer (alcohol) + (−) − enantiomer(alcohol); ²⁾the percentage ee value was determined in accordance withthe following equation: % excess enantiomer − % deficit enantiomer/%excess enantiomer + % deficit enantiomer.

Example 4

Enzymatic Ester Hydrolysis

Lipase-catalysed hydrolysis of (1SR,3RS,4RS)-butyric acid3-dimethylaminomethyl-4-hydroxy-4-(3-methoxy-phenyl)-cyclohexyl ester(rac-1)

(+)-(1S,3R,4R)-Butyric acid3-dimethylaminomethyl-4-hydroxy-4-(3-methoxy-phenyl)-cyclohexyl ester((+)-1)and(−)-(1S,2S,4R)-2-Dimethylaminomethyl-1-(3-methoxy-phenyl)-cyclohexane-1,4-diol((−)-2)

Analogously to that described in example 3, enzymatic hydrolysis ofrac-1 using the lipase Candida rugosa (Fluka) in an aqueous buffersystem at a pH of 7.5 using 10% tert-butanol leads to an oppositeasymmetric induction after a reaction time of 24 hours at roomtemperature. After a conversion of 28%, the alcohol (−)-2 could beisolated with an ee value of 89% and the ester (+)-1 with an ee value of37% (E=24).

Example 5

Enzymatic Ester Hydrolysis

Pig liver esterase-catalysed hydrolysis of (1SR,3RS,4RS)-butyric acid4-dimethylaminomethyl-3-hydroxy-3-(3-methoxy-phenyl)-cyclohexyl esterhydrochloride (rac-3)

(+)-(1R,3S,6R)-6-Dimethylaminomethyl-1-(3-methoxy-phenyl)-cyclohexane-1,3-diol((+)-4)and(−)-(1R,3S,4S)-Butyric acid4-dimethylaminomethyl-3-hydroxy-3-(3-methoxy-phenyl)-cyclohexyl ester((−)-3)

Analogously to that described in example 3, enzymatic hydrolysis ofrac-3 using pig liver esterase in an aqueous buffer system at a pH of8.0 using 10% tert-butanol leads to a conversion of 40% after a reactiontime of 6 hours at room temperature. It was possible in this manner toobtain the ester (−)-3 in a yield of 79% with an ee value of 86%([α]_(D) ²²=−6.0° (c=0.81, methanol)) and the alcohol (+)-4 in a yieldof 77% with an ee value of 94% ([α]_(D) ²²=+21.7° (c=0.80, methanol))(E=46).

Example 6

Enzymatic Ester Hydrolysis

Lipase-catalysed hydrolysis of (1SR,3RS,4RS)-butyric acid4-dimethylaminomethyl-3-hydroxy-3-(3-methoxy-phenyl)-cyclohexyl esterhydrochloride (rac-3)

(−)-(1S,3R,6S)-6-Dimethylaminomethyl-1-(3-methoxy-phenyl)-cyclohexane-1,3-diol((−)-4)and(+)-(1S,3R,4R)-Butyric acid4-dimethylaminomethyl-3-hydroxy-3-(3-methoxy-phenyl)-cyclohexyl ester((+)-3)

Analogously to that described in example 3, enzymatic hydrolysis ofrac-3 using the lipase Candida rugosa in an aqueous buffer system at apH of 7.0 using 10% tert-butyl methyl ether leads to a conversion of 45%after a reaction time of 6 hours at room temperature. It was possible inthis manner to obtain the eater (+)-3 in a yield of 80% with an ee valueof >99% ([α]_(D) ²²=+7.5° (c=0.74, methanol)) and the alcohol (−)-4 in ayield of 79% with an ee value of >99% ([α]_(D) ²²=−29.5° (c=1.01,methanol)) (E>200).

Example 7

Enzymatic Transacylation in Organic Solvents

Lipase-catalysed transacylation of(1RS,3SR,6RS)-6-dimethylaminomethyl-1-(3-methoxy-phenyl)-cyclohexane-1,3-diolrac-4 with various acylating reagents to give the esters 5 and 6

(1R,3S,6R)-6-Dimethylaminomethyl-1-(3-methoxy-phenyl)-cyclohexane-1,3-diol(+)-4andR⁵=CH₃: (−)-(1R,3S,4S)-Acetic acid4-dimethylaminomethyl-3-hydroxy-3-(3-methoxy-phenyl)-cyclohexyl ester(−)-5or

-   R⁵=CH₂CH₃; (−)-(1R,3S,4S)-Propionic acid    4-dimethylaminomethyl-3-hydroxy-3-(3-methoxy-phenyl)-cyclohexyl    ester (−)-6

For the transacylation, 70 mg (0.25 mmol)(1RS,3SR,6RS)-6-dimethylaminomethyl-1-(3-methoxy-phenyl)-cyclohexane-1,3-diolrac-4 were taken up in a solvent mixture comprising toluene and thetransacylating reagent or using the transacylating agent itself as thesolvent, and the mixture was first stirred at room temperature for twohours. After addition of the lipase Candida rugosa (5 mg, 185 units),the mixture was stirred at room temperature for 5 to 9 days. To separateoff the enzyme, the mixture was filtered over silica gel. The alcoholand ester were separated from one another as described in example 1 andisolated. The results are summarized in table 4.

Instead of the lipase Candida rugosa, the lipases Candida cylindracea orPseudomonas cepacia were also employed in an analogous manner.

TABLE 4 Results of the enzymatic transacylation of(1RS,3SR,SRS)-6-dimethylaminomethyl-1-(3-methoxy-phenyl)-cyclohexane-1,3-diol rac-4 Reaction % ee % ee valueExample Transacylating time Conversion value of of the E no. reagent,solvent (days) (%) Ester the ester Alcohol alcohol value 5a

9 48 (−)-6 87 (+)-4 68 30 5b

7 56 (−)-5 91 (+)-4 97 89 5c

5 34 (−)-5 99 (+)-4 60 >200Nomenclature Overview

Formula Nomenclature

(1RS,2RS,4SR)-2-Dimethylaminomethyl-1-(3-methoxy-phenyl)-cyclohexane-1,4-dial

(1RS,3RS,6RS)-6-Dimethylaminomethyl-1-(3-methoxy-phenyl)-cyclohexane-1,3-diol

(1RS,3SR,6RS)-6-Dimethylaminomethyl-1-(3-methoxy-phenyl)-cyclohexane-1,3-diol

(1SR,3RS,4RS)-Butyricacid3-dimethylaminomethyl-4-hydroxy-4-(3-methoxy-phenyl)-cyclohexylester

(1SR,3RS,4RS)-Butyricacid4-dimethylaminomethyl-3-hydroxy-3-(3-methoxy-phenyl)-cyclohexylester

(−)-(1R,3S,4S)-Aceticacid4-dimethylaminomethyl-3-hydroxy-3-(3-methoxy-phenyl)-cyclohexylester

(−)-(1R,3S,4S)-Propionicacid4-dimethylaminomethyl-3-hydroxy-3-(3-methoxy-phenyl)-cyclohexyl ester

1. A process for the separating enantiomers ofaminomethyl-aryl-cyclohexanol compounds corresponding to formula I

wherein X is chosen from H, F, Cl, Br, I, CF₃, O—S(O₂)—C₆H₄-pCH₃, OR¹⁴or OC(O)R¹⁴, wherein R¹⁴ is chosen from H; C₁–C₁₀-alkyl, C₂–C₁₀-alkenylor C₂–C₁₀-alkinyl, in each case branched or unbranched and mono- orpolysubstituted or unsubstituted; C₃–C₇-cycloalkyl, saturated orunsaturated and mono- or polysubstituted or unsubstituted, or acorresponding heterocyclic radical in which a C atom in the ring isreplaced by N, S or O; alkylaryl or alkylheteroaryl, saturated orunsaturated and mono- or polysubstituted or unsubstituted; aryl orheteroaryl, in each case mono- or polysubstituted or unsubstituted; R³,R⁴ independently of one another are chosen from H, C₁–C₁₀-alkyl,C₂–C₁₀-alkenyl or C₂–C₁₀-alkinyl, in each case branched or unbranchedand mono- or polysubstituted or unsubstituted; C₃–C₇-cycloalkyl,saturated or unsaturated and mono- or polysubstituted or unsubstituted,or a corresponding heterocyclic radical in which a C atom in the ring isreplaced by N, S or O; alkylaryl or alkylheteroaryl, saturated orunsaturated and mono- or polysubstituted or unsubstituted; aryl orheteroaryl, in each case mono- or polysubstituted or unsubstituted; orR³ and R⁴ together form a C₃–C₇-cycloalkyl, saturated or unsaturated andmono- or polysubstituted or unsubstituted, or a correspondingheterocyclic radical in which a C atom in the ring is replaced by S, Oor NR¹⁵, where R¹⁵ is chosen from H, C₁–C₁₀-alkyl, C₂–C₁₀-alkenyl orC₂–C₁₀-alkinyl, in each case branched or unbranched and mono- orpolysubstituted or unsubstituted; R¹ and R² independently of one anotherare either H or any desired substituent and in each case one of thesubstituents R⁵ and R⁶ corresponds to H and the other corresponds to OH,characterized in that, depending on the desired enantiomer of theaminomethyl-aryl-cyclohexanol compounds corresponding to formula Icomprising, according to reaction I, esterifying compounds correspondingto formula I and enzymatically transforming the compounds and separatingenantiomerically pure compounds or comprising, according to reaction II,enzymatically transforming compounds corresponding to formula I in thepresence of an ester and separating enantiomerically pure compounds. 2.A process according to claim 1, comprising in reaction I, enzymaticallytransforming a compound corresponding to formula II

in which the substituent OC(O)R⁷ corresponds to the position of R⁵ or R⁶in formula I and R⁷ is chosen from C₁–C₆-alkyl, unsubstituted or mono-or polysubstituted; as the free base or in the form of its salt, in asolvent with a lipase or esterase and separating enantiomerically purecompounds corresponding to formulae III and Ia

where compounds according to formula Ia correspond to compoundsaccording to formula I and the substituent OH corresponds to theposition of R⁵ or R⁶ in formula I.
 3. The process according to claim 2,characterized in that R⁷ is chloroacetyl, butyl or pentyl.
 4. Theprocess according to claim 2, characterized in that the enzyme used isan esterase.
 5. The process according to claim 2, characterized in thatan aqueous buffer system is used as the solvent.
 6. The processaccording to claim 2, characterized in that an aqueous buffer system,preferably with a physiological pH for the enzyme used, is used as thesolvent.
 7. The process according to claim 5, characterized in that atleast one organic solvent is added to the aqueous buffer system up to apercentage content by volume of between 1 and 50%.
 8. The processaccording to claim 2, characterized in that the compound according toformula II is employed as the hydrochloride salt.
 9. The processaccording to claim 2, characterized in that the compounds according toformula II employed are prepared by a process in which racemic compoundsaccording to formula I

are converted with bases in a solvent into the alcoholates andsubsequently, with the addition of corresponding acid halides, into theracemic esters according to formula II

in which the substituent OC(O)R⁷ corresponds to the position of R⁵ or R⁶in formula I.
 10. The process according to claim 1, comprising, inreaction alternative II, enzymatically transforming a racemic compoundcorresponding to formula I

employed as the free base or in the form of its salt in a solvent withan ester according to formula IV

wherein, independently of one another, R⁸ denotes C₁–C₆alkyl,substituted or unsubstituted; and R⁹ denotes H or C₁–C₆-alkyl,substituted or unsubstituted, with a lipase or esterase and separatingthe enantiomerically pure compounds formed, corresponding to formulae Vand Ib

wherein compounds according to formula Ib correspond to compoundsaccording to formula I and the substituent OH corresponds to theposition of R⁵ or R⁶ in formula I.
 11. The process according to claim10, characterized in that in the esters according to formulae IV and V,R⁸ denotes methyl or ethyl or R⁹ according to formula IV denotes H ormethyl.
 12. The process according to claim 10, characterized in that theester according to formula IV is vinyl propionate, vinyl acetate orisopropenyl acetate.
 13. The process according to claim 10,characterized in that the enzyme used is a lipase.
 14. The processaccording to claim 10, characterized in that an organic solvent is usedas the solvent.
 15. The process according to claim 1, characterized inthat ester/alcohol mixtures are separated by pH-selective extractionafter conclusion of the enzymatic transformation.
 16. The processaccording to claim 1, characterized in that R¹ and R² in the formulae I,Ia, Ib, II, III and V independently of one another are chosen from R¹⁰or YR¹⁰, where Y=C₁–C₁₀-alkyl, C₂–C₁₀-alkenyl or C₂–C₁₀-alkinyl,branched or unbranched and mono- or polysubstituted or unsubstituted,wherein R¹⁰ is chosen from H, F, Cl, Br, I, CN, NO₂, C₁–C₈-alkyl,C₂–C₈-alkenyl or C₂–C₈-alkinyl, in each case branched or unbranched andmono- or polysubstituted or unsubstituted; C₃–C₇-cycloalkyl, saturatedor unsaturated and mono- or polysubstituted or unsubstituted, or acorresponding heterocyclic radical in which a C atom in the ring isreplaced by S, O or N; aryl or heteroaryl, in each case mono- orpolysubstituted or unsubstituted; OR¹¹, OC(O)R¹¹, OC(O)OR¹¹, OC(S)R¹¹,C(O)R¹¹, C(O)OR¹¹, C(S)R¹¹, C(S)OR¹¹, SR¹¹, S(O)R¹¹ or S(O₂)R¹¹, whereinR¹¹ is chosen from H, C₁–C₁₈-alkyl, C₂–C₁₈-alkenyl or C₂–C₁₈-alkinyl, ineach case branched or unbranched and mono- or polysubstituted orunsubstituted; C₃–C₇-cycloalkyl, saturated or unsaturated and mono- orpolysubstituted or unsubstituted, or a corresponding heterocyclicradical in which a C atom in the ring is replaced by S, O or N;alkylaryl or alkylheteroaryl, saturated or unsaturated and mono- orpolysubstituted or unsubstituted; aryl or heteroaryl, in each case mono-or polysubstituted or unsubstituted; or NR¹²R¹³, C(O)NR¹²R¹³ orS(O₂)NR¹²R¹³, wherein R¹² and R¹³ independently of one another arechosen from H, C₁–C₁₈-alkyl, C₂–C₁₈-alkenyl or C₂–C₁₈-alkinyl, in eachcase branched or unbranched and mono- or polysubstituted orunsubstituted; C₃–C₇-cycloalkyl, saturated or unsaturated and mono- orpolysubstituted or unsubstituted, or a corresponding heterocyclicradical in which a C atom in the ring is replaced by S, O or N;alkylaryl or alkylheteroaryl, saturated or unsaturated and mono- orpolysubstituted or unsubstituted; aryl or heteroaryl, in each case mono-or polysubstituted or unsubstituted; or R¹² and R¹³ together form aC₃–C₇-cycloalkyl, saturated or unsaturated and mono- or polysubstitutedor unsubstituted, or a corresponding heterocyclic radical in which a Catom in the ring is replaced by S, O or N; or R¹ and R² together form—CH═CH—CH═CH—, wherein the naphthyl system formed can be mono- orpolysubstituted.
 17. The process according to claim 1, characterized inthat R¹=R¹⁰, wherein R¹⁰ is chosen from H, F, Cl, Br, I, CF₃, NO₂, NH₂;C₁–C₄-alkyl or C₂–C₄-alkenyl, branched or unbranched and mono- orpolysubstituted or unsubstituted; OR¹¹, C(O)OR¹¹ or SR¹¹, wherein R¹¹ ischosen from H; C₁–C₄-alkyl, branched or unbranched and mono- orpolysubstituted or unsubstituted; preferably H, CF₃ or CH₃, orS(O₂)NR¹²R¹³, wherein R¹² and R¹³ independently of one another arechosen from H; C₁–C₄-alkyl, branched or unbranched and mono- orpolysubstituted or unsubstituted.
 18. The process according to claims 1,characterized in that R²=R¹⁰, wherein R¹⁰ is chosen from H, F, Cl, Br,I, SCH₃; C₁–C₄-alkyl, C₂–C₄-alkenyl, branched or unbranched and mono- orpolysubstituted or unsubstituted, preferably CF₃; OR¹¹, where R¹¹ ischosen from C₁–C₄-alkyl, branched or unbranched and mono- orpolysubstituted or unsubstituted, preferably CH₃.
 19. The processaccording to claim 1, characterized in that X is chosen from H, F, Cl,OH, CF₃, O—S(O₂)—C₆H₄-pCH₃ or OC(O)R¹² where R¹²=H; C₁–C₄-alkyl orC₂–C₄-alkenyl, branched or unbranched and mono- or polysubstituted orunsubstituted, preferably H, F, Cl, OH, O—S(O₂)—C₆H₄-pCH₃, OC(O)R¹²where R¹²=C₁–C₄-alkyl, preferably CH₃.
 20. The process according toclaim 4, wherein the enzyme used is a pig liver esterase.
 21. Theprocess according to claim 5, wherein the solvent is an aqueous buffersystem, with a pH of between 6.0 and 8.0.
 22. The process according toclaim 7, wherein the organic solvent is acetone or butanol.
 23. Theprocess according to claim 7, characterized in that at least one organicsolvent is added to the aqueous buffer system up a percentage content byvolume of between 5 and 20%.
 24. The process according to claim 9,wherein the bases include potassium tert-butylate or sodium hyride. 25.The process according to claim 9, wherein the solvent is tetrahydrofuranor dimethylformamide.
 26. The process according to claim 13, wherein thelipase is a lipase from Candida rugosa, Candida cylindracea orPseudomonas cepacia.
 27. The process according to claim 14, wherein theorganic solvent is toluene.
 28. The process according to claim 17,wherein R¹ is chosen from H, F, Cl, OH, CH₃, C₂H₅, C₂H₃, CF₃, SCH₃,OCF₃, OCH₃, OC₂H₅, C(O)OCH₃, C(O)OC₂H₅.
 29. The process according toclaim 17, wherein R¹ is m-OCH₃.
 30. The process according to claim 18,wherein R² is H.
 31. The process according to claim 19, wherein X is OH,F or Cl.